Thymidine-synchronized cells

Synchronizing Cell Cultures - Laborotory procedure of manipulating cells to bring all of the them to the same phase of the cell cycle. It is accomplished by a thymidine block. 

Thymidine Block - Mechanism for synchronizing cells in which thymidine is added to cells to inhibit DNA synthesis. This prevents passage of the cells from the G1 to S phase of the cell cycle. Subsequent transfer of cells to a medium lacking thymidine reverses the inhibition and allows them to initiate DNA synthesis synchronously. 

The buoyant densities of replicating DNA in different portions of S phase have been studied in thymidine-synchronized HeLa and mouse L-cells (Tobia et al., 1970). Early in S, DNA molecules having high buoyant densities were more readily radioactively labeled,... 

In conclusion, an inhibitor of thymidylate synthesis and uptake and thus of DNA synthesis has been found. It consists of two components, methotrexate and uridine. It inhibits DNA synthesis to the extent and in ways which lead to blockage of the next cell division. This would be expected if DNA replication itself or events coupled to progression in the S phase are required to take the cell on to division. Inhibition by methotrexate + uridine can be discontinued by addition of excess thymidine, or just by washing of the cells with fresh growth medium, with or without added folate. These agents shall be used as tools in attempts to control DNA replication independently of cell division in populations with temperature-synchronized cell division. 

Up to now we have assumed that cells in S are immediately stopped by methotrexate + uridine, and that cells not in S when this inhibitor is present continuously collect just before S. It is on this basis that we can assume that the time from the addition of thymidine to synchronous cell division is a measure of the interval S + G2 + 1/2 D. A second tacit assumption is that cells treated with methotrexate + uridine for some time can be made to progress in the cycle at the normal rate when supplied with thymidine. Neither of the two assumptions may be fully correct, but fortunately errors involved are likely to cancel out. Concerning the first assumption, there is in fact evidence that cells treated with methotrexate + uridine may progress perhaps 10 minutes into S before they are arrested (see Table 5 in Zeuthen, 1968) which might fit the observations by Stone et al. (1965) that acid-soluble thymidine compounds are carried over in the macronucleus from one S period to the next and are used for DNA synthesis. In any case, such effects... 

Fig. 24a. Inhibition of synchronized cell divisions (system of Scherbaum and Zeuthen, 1954) with combinations of 5-fluoro>2 deoxyuridine (FUdR), uridine (U), and thymidine (T). b. Same, only 5-fluorouridine (FUR) is used.

Figure 3. Scanning electron micrographs of synchronized KB cells at various stages of cell cycle. Cells pretreated for 20 hr with 2 mM thymidine, released for 8 hr, and mitotically selected by shaking, (a) Mitotic cells (X200) (b) early G, phase (X800) (c) late G,-early S phase (X1200) (d) S phase (X2000).

As a result, S phase was prolonged in these cells (Figure 2). Following G1 treatment with cisplatin, synchronized Hela cells also showed a decrease in the amount of thymidine incorporation into DNA during S phase, but the effect was not immediately manifested in these cells (63) (Figure 3). Thus cells differ in the way in which their replication machinery responds to cispla-tin-induced daimage. Such differences might account for variations in cellular sensitivity (see below) and further studies in this area are warranted. 

Fig. (8). Inhibitory eflect of magnoshinin and magnosalin, neolignanes derived from "shin-i" (Flos Magnoliae) on proliferation of subcultured rat aortic endothelial cells (ECs) stimulated with 5% fetal bovine serum (FBS). Subconfluent ECs (cultured for 6 days) were synchronized by serum starvation for 2 days, and stimulated with 5% FBS and [ H]>thymidine in the presence and absence of the above compounds (0.1, 0.3, I or 3 pg/mL). Tritium thymidine incorporation was measured at 3-hr intervals in culture. The values represent means S.E.M. (n = 3-9).

Fig. (9 b). Inhibitory effects of BP-42, BP-421 and BP-422 on the PDGF-BB-induced competence phase of [ H]-thymidine incorporation in primary cultured and synchronized rat aorta SMCs. The SMCs were incubated for 3 hr with PDGF-BB (30 ng/mL) plus each BP compound ( 0-10 ig/mL), and with 10% FBS alone ( o). After washing out, the cells were incubated with [ H]-thymidine in the presence of 1% FBS ( ) and 10% FBS (o). The values represent means S.E.M. (n = 5-8). 

In HeLa ceils hydroxyurea is an efficient inhibitor of histone synthesis. This action requires protein synthesis and leads to rapid disappearance of cytoplasmatic histone mRNA The effect is not specific for hydroxyurea since suppression of DNA synthesis by arabino-cytosine or temperature-sensitive mutations yields analogous results. Similarly, the synthesis of some enzymes necessary for DNA replication and active in S-phase is altered by hydroxyurea. Increased activity of ribonucleotide reductase in HeLa and in hamster cells and of the salvage enzyme thymidine kinase in HeLa cells and KB cells has been observed, probably as a consequence of the increased fraction of cells in S-phase. Repression occurs for thymidine kinase in human lymphocytes and for ornithine decarboxylase in Chinese hamster fibroblasts whereas no or only slight effects were seen on ribonucleotide reductase in hamster fibroblasts , on thymidylate synthase in extracts from synchronous mouse cells " , and on DNA polymerase in rabbit kidney cells or HeLa cells . ... 

Fig. 2. DNA synthesis in the cell cycle of mouse L-cells. Mouse L-cells were synchronized by two treatments with 2.5 x thymidine for 16 hours, separated by a 9-hour period in the absence of...

Density labeling procedures have been used along with synchronously growing populations of cells to study DNA synthesis in subfractions of the S phase in several eukaryote systems. In Physarum polycephalum, Braun et al. (1965) were able to radioactively label replicating DNA in the second half of one S i ase with tritiated thymidine and then density label all subsequent replicating DNA by the addition of bromodeoxyuridine to the cultures. Analysis in cesium chloride equilibrium gradients of DNA radioactively labeled, and then density labeled in this manner, indicated that DNA... 

Some of the chromosomal regions boxmd to the nuclear membrane may be the sites where DNA synthesis is initiated (Comings and Kakefuda, 1968). When the site of incorporation of radioactive thymidine was studied at the start of the S phase in synchronized human amnion cell cultures, the label was found to be localized at the nuclear membranes (Comings and Kakefuda, 1968), suggesting that the chromosomal sites at which DNA synthesis was initiated were attached to the nuclear membrane. It was... 

Fig. 3. Curve A, Percentage nuclei with labeled DNA when the synchronized population is incubated with thymidine from EH. Curve B, Percent labeled in 5-minute pulses placed at various times after EH. The times indicate the terminations of the pulses. (From Hjelm and Zeuthen. 1967b. Exp. Cell / es., 48 231-232.)...

Figure 7 shows that a synchronous division occurs between 110 and 120 minutes after thymidine is added. The first cells to divide are assumed to be those which have been stopped in late S by methotrexate + uridine. When thymidine is added they quickly finish the S period, pass through G2, and divide. Thus, the time 110 to 120 minutes is a G2 + D period. It is extended as compared with the normal value which is 91 minutes (Table 3). So, contrary to what is observed for the S interval, the G2 + D interval appears to be extended when DNA synthesis is synchronized by starvation for, and new feeding of, thymine compounds. 

Control experiments showed that synchronous divisions, as reflected in changes of the division index, can be induced normally with heat shocks in the presence of either uridine, thymidine, or methotrexate. With uridine or with thymidine added before the shocks the pattern of synchronous divisions is similar to what is seen in the control, but with methotrexate the first division is slightly delayed and the second division much delayed, resembling what was observed with exponentially multiplying cells. On the other hand, when the inhibitor complex of DNA synthesis, methotrexate + uridine, is added just before the seven heat shocks, all later division activity is practially eliminated. When it is added during the period of heat shocks, more and more cells will take part in division 1 the later the agent is supphed. When the inhibitor is added in the interval between shocks 6 and 7 there is extensive participation in the first division, but no cells ever divide a second time. When the inhibitor is added at EH7 there is nearly full participation in division 1, and a few calls can take part also in the second division. From such... 

Fig. 9. Synchronization with seven heat shocks of two parallel 150-ml cultures, of which one served as a control (C). The second received methotrexate + uridine (M + U) after shock 3. After washing of both populations with fresh medium after heat shock 7, 0.05-ml aliquots were drawn from both flasks. They were pulsed for 10 minutes with tritiated thymidine, and 10 to 20=jLil samples were fixed for autoradiography. The percentage of macronuclei labeled in acid-stable compounds (DNA) were plotted at the end of the incorporation intervals (lower curves). The upper curves show the percentage of dividers against time. The control goes from 0 to 95 percent in division 1 (visual estimates). (From Zeuthen. 1970. Exp. Cell Res., 61 311 -325.)...

This experiment supplies the additional information that the first, and in turn the second synchronous division can be delayed by a shortage of thymidine, and for a time equal to the delay beyond the time CTi by which methotrexate + uridine-treated cells received thymidine. Thus, this experiment supplies no evidence of damage to the system by methotrexate + uridine once these agents have collected the cells at a point just before S. Possibly cells are damaged when methotrexate + uridine is supplied in the replication phase. 

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